High-resolution melting analysis

Abstract

The present invention relates to methods and systems for the analysis of the dissociation behavior of nucleic acids and the identification of nucleic acids. In one aspect, methods and systems are disclosed for resolving a denaturation curve of a sample containing a first and second nucleic acid into a resolved denaturation curve for the first nucleic acid and a resolved denuration curve for the second nucleic acid.

1. A method of resolving a denaturation curve of a sample containing a first and second nucleic acid into a resolved denaturation curve for the first nucleic acid and a resolved denaturation curve for the second nucleic acid, said method comprising:
 (a) deriving a denaturation curve from denaturation data; 
 (b) calculating an estimated intrinsic physical value associated with the first nucleic acid by using data from a non-overlapping region of the denaturation curve; 
 (c) calculating a first resolved denaturation curve for the first nucleic acid using the estimated intrinsic physical value; and 
 (d) subtracting the first resolved denaturation curve for the first nucleic acid from the denaturation curve to generate a second resolved denaturation curve for the second nucleic acid. 
 
     
     
       2. The method of  claim 1 , wherein the denaturation data includes measurements of a quantifiable physical change P of the sample at a plurality of independent sample property points x, wherein the quantifiable physical change P is associated with denaturation of a nucleic acid. 
     
     
       3. The method of  claim 2 , wherein the plurality of independent sample property points x are a plurality of temperatures. 
     
     
       4. The method of  claim 3 , wherein the intrinsic physical property is the enthalpy of denaturation of the first nucleic acid. 
     
     
       5. The method of  claim 4 , further comprising calculating the van't Hoff enthalpy of the first nucleic acid by using data from the non-overlapping region of the denaturation curve. 
     
     
       6. The method according to  claim 5 , further comprising the step of determining an estimated van't Hoff enthalpy and entropy change of the second nucleic acid from the resolved denaturation curve for the second nucleic acid. 
     
     
       7. The method according to  claim 1 , further comprising the step of determining a melting temperature of the first nucleic acid from the resolved melting curve for the first nucleic acid. 
     
     
       8. The method of  claim 1 , further comprising the step of determining a melting temperature of the second nucleic acid from the resolved denaturation curve for the second nucleic acid. 
     
     
       9. The method according to  claim 2 , wherein said sample further includes a double-strand specific fluorescent dye, and wherein said quantifiable physical change is the fluorescence intensity. 
     
     
       10. The method according to  claim 1 , wherein the first nucleic acid and second nucleic acid are single nucleotide polymorphisms of one another. 
     
     
       11. The method according to  claim 1  further comprising the step of generating denaturation data including measurements of a quantifiable physical change P of the sample at a plurality of independent sample property points x for a sample, wherein the quantifiable physical change P is associated with denaturation of a nucleic acid. 
     
     
       12. The method according to  claim 1 , wherein the denaturation data is thermal melt data. 
     
     
       13. A system for resolving a denaturation curve of a sample containing a first nucleic acid and a second nucleic acid into a resolved melt curve for the first nucleic acid and a resolved melt curve for the second nucleic acid, said system comprising:
 (a) a plotting module capable of plotting a denaturation curve from denaturation data; 
 (b) an estimating module capable of calculating an estimated intrinsic physical value associated with the first nucleic acid by using data from a non-overlapping region of a denaturation curve; and 
 (c) a resolving module capable of calculating a resolved denaturation curve for the first nucleic acid using the estimated intrinsic physical value, and subtracting the first resolved denaturation curve for the first nucleic acid from a denaturation curve to generate a second resolved denaturation curve for the second nucleic acid. 
 
     
     
       14. The system of  claim 13 , wherein the denaturation data includes measurements of a quantifiable physical change P of the sample at a plurality of independent sample property points x, wherein the quantifiable physical change P is associated with the denaturation of a nucleic acid. 
     
     
       15. The system of  claim 14 , wherein said estimating module is further capable of calculating the van't Hoff enthalpy of the first nucleic acid by using data from the non-overlapping region of the denaturation curve. 
     
     
       16. The system of  claim 15 , further comprising a thermodynamic information determining module capable of determining an estimated van't Hoff enthalpy and entropy change of the second nucleic acid from the second resolved denaturation curve for the second nucleic acid. 
     
     
       17. The system of  claim 13 , further comprising a melting temperature determining module capable of determining the melting temperature of the first nucleic acid from the resolved denaturation curve for the first nucleic acid. 
     
     
       18. The system of  claim 13 , further comprising a melting temperature determining module capable of determining the melting temperature of the second nucleic acid from the resolved denaturation curve for the second nucleic acid.